YOLK STEROIDS DECLINE DURING SEXUAL-DIFFERENTIATION IN THE ALLIGATOR

The leading explanation of temperature-dependent sex determination (TS D) in reptiles postulates that (1) ovarian differentiation is directed by estrogen and that (2) estrogen is synthesized in the developing go nad following induction of aromatase expression. However, the source o f steroid substrate for aromatization has not yet been identified. In addition, sex ratios vary as a function of clutch, but such biases are as yet unexplained. To address these issues, we measured estradiol, t estosterone, and androstenedione in yolks of the American alligator (A lligator mississippiensis) before, during, and after the period of gon adal differentiation in this TSD species. Eggs were collected from a w ild population in Louisianna and were incubated at male- and female-de termining constant temperatures in the lab, as well as at intermediate temperatures that produced both sexes. Steroids were assayed in yolk extracts after celite column chromotography. All three steroids were f ound to be in the range of nanograms/gram of yolk at stage 16. Androst enedione was the predominant steroid, 2- to 3-fold higher in concentra tion than estradiol and 15- to 20-fold higher than testosterone. The l evels of these steroids declined (5- to 30-fold) between stages 16 and 25, most markedly between stages 21 and 23, regardless of incubation temperature. The chronology of this sharp decline in steroid levels in our study coincides with the timing of gonadal differentiation in thi s species, between stages 21 to 23 based on previous reports. Estradio l levels in yolks differed by 3-fold in some clutches relative to othe rs, whereas, no clutch differences were apparent for either androstene dione or testosterone. These data demonstrate that alligator yolk cont ains high concentrations of two steroid substrates utilized for estrog en synthesis, as well as significant quantities of estradiol itself. W e hypothesize that estradiol levels in yolk provide a steroid backgrou nd, variable among and within clutches, on which gonadal development i s initiated and proceeds. As a consequence, we suggest that yolk provi des an epigenetic maternal contribution that modulates the effect of i ncubation temperature on hatchling sex. (C) 1997 Academic Press.